Yeast-based HTS Assay Technologies for Proteases. Proteolytic processing of proteins is an irreversible post-translational modification of importance for a wide-variety of biological processes. Consequently, proteases have emerged as promising targets for drug discovery for a wide variety of human diseases, including inflammation, infectious diseases, neurodegeneration, ischemic diseases, and cancer. Development of high throughput screening (HTS) assays using purified proteases can be relatively straightforward or it can be quite challenging, particularly when multi-component systems are required to achieve protease activation. Also, due to similarity of the active sites of some groups of proteases, selectivity of chemical inhibitors can be difficult if not impossible to achieve, highlighting the need for alternative screening methods for identifying compounds that target upstream activators of proteases rather than directly inhibiting the protease of interest. We propose to generate and optimize HTS systems for intracellular proteases, using Caspases as a prototype. For this purpose, we have devised yeast-based cellular systems that permit facile expression of proteases and protease-activating proteins in combinations that reconstitute entire mammalian pathways in these simple eukaryotes. Among the assay methods integrated into the yeast system are cleavable reporter gene activators, in which protease-mediated cleavage activates a transcription factor. The Aims are to: (1) Devise multi-component systems that reconstitute mammalian protease activation pathways in yeast;(2) Adjust the necessary variables to achieve HTS-quality assay performance;(3) Perform pilot chemical library screens of multi-component yeast-based protease assay systems to define hit-rates and test reliability;and (4) Develop secondary assay strategies and methods for post- screening hit deconvolution and validation. In addition, we will validate this HTS technology by applying it for a full-fledged HTS campaign in which compounds will be identified and optimized that selectively inhibit the upstream Caspase-1 activator NLRC4 (Ipaf1;CLAN), a component of innate immunity and critical regulator of host responses to intracellular bacterial pathogens. PUBLIC HEALTH RELEVANCE: Proteases are proteins that cleave other proteins. These enzymes play important roles in many diseases. Consequently, proteases have emerged as promising targets for drug discovery, but it can often be challenging to obtain selective inhibitors. We propose to devise a novel technology for high- throughput screening of large collections of chemicals for identifying chemical modulators of the upstream activators of intracellular proteases. For proof of concept, we focus on proteases important for inflammatory and infectious diseases.